The present invention relates to: manipulation of the amino acid sequence of tropoelastin, particularly human tropoelastin, to modify its protease susceptibility; to tropoelastin derivatives having modified protease susceptibility; to peptidomimetic molecules which contain amino acid sequences which correspond to or incorporate the protease susceptible sequences of tropoelastin; and to uses of the tropoelastin derivatives and peptidomimetic molecules.
The invention also relates to nucleic acid molecules and genetic constructs encoding the amino acid sequences of the derivatives and peptidomimetic molecules of the invention.
The insoluble cross-linked elastin molecule is highly resistant to proteolytic degradation by many proteases. However, tropoelastin, the soluble precursor of elastin, is far more vulnerable to proteolysis. Attempts at purifying tropoelastin from tissues usually result in a collection of degraded products. This degradation can be decreased by using traditional inhibitors of serine proteases (Franzblau et al., 1989; Rucker, 1982, Rich and Foster, 1984; Sandberg and Wolt, 1982). Specific degradation has also been noted in cell culture of smooth muscle cells which was attributed to metalloproteinases (Hayashi et al., 1995). Even highly purified tropoelastin can degrade into discrete bands on prolonged storage. This observation has led to a hypothesis that mammalian tropoelastin is occasionally co-purified with an intrinsic protease which will promote its gradual breakdown (Mecham et al., 1976; Mecham et al., 1977; Mecham and Foster, 1977). Experiments have shown that mammalian serum contains proteases which are capable of degrading tropoelastin (Romero et al., 1986). Thus, any newly-synthesized unprotected tropoelastin exposed to blood, such as in a blood vessel wall, would be rapidly degraded. Serum has also been shown to induce elastase activity in smooth muscle cells leading to degradation of elastin (Kobayashi et al., 1994). Elastin peptides are known to be chemotactic and this could be a role of tropoelastin proteolysis in vivo (Grosso and Scott, 1993; Bisaccia et al., 1994). However, proteolysis could also result in inadequate or faulty elastin fiber repair at the site of injury. Serine protease inhibitors have been shown to reduce the degradation of tropoelastin caused by serum (Romero, et al., 1986). These experiments suggested that kallikrein was a candidate serum protease. Other experiments (McGowan et al., 1996) proposed that plasmin was a major protease involved. Thrombin has been used to digest heterogeneous porcine tropoelastin in vitro (Torres et al., 1976). However, none of these studies has provided indication of where the tropoelastin molecule is cut by proteases.